Colorectal cancer is a major public health issue worldwide. Interleukin-17 (IL-17) and Th17 (T-helper cell type 17)-related molecules are involved in tumor development and in resistance to bevacizumab, an anti-vascular endothelial growth factor monoclonal antibody used in association with chemotherapy in metastatic colorectal cancer.
Lereclus et al BMC Cancer (2017) 17:220 DOI 10.1186/s12885-017-3210-z RESEARCH ARTICLE Open Access A possible association of baseline serum IL-17A concentrations with progression-free survival of metastatic colorectal cancer patients treated with a bevacizumab-based regimen Emilie Lereclus1†, Mira Tout1†, Alban Girault1, Nadine Baroukh1, Morgane Caulet1,2, Christophe Borg4, Olivier Bouché5, David Ternant1,3, Gilles Paintaud1,3, Thierry Lecomte1,2 and William Raoul1* Abstract Background: Colorectal cancer is a major public health issue worldwide Interleukin-17 (IL-17) and Th17 (T-helper cell type 17)-related molecules are involved in tumor development and in resistance to bevacizumab, an anti-vascular endothelial growth factor monoclonal antibody used in association with chemotherapy in metastatic colorectal cancer Some studies have previously shown that IL-17A and IL-17F polymorphisms, respectively rs2275913 and rs763780, are associated with gastric or colorectal cancer risk Here we aimed at studying the influence of IL-17A-related individual factors on overall survival and progression-free survival in patients with metastatic colorectal cancer treated with a bevacizumab-based chemotherapy Methods: Pre-treatment serum biomarkers were retrospectively evaluated in 122 metastatic colorectal cancer patients treated by bevacizumab in combination with chemotherapy at 2-weeks intervals in a prospective cohort study (NCT00489697) The polymorphisms of IL-17A and IL-17F were analyzed by polymerase chain reaction - restriction fragment length polymorphism Serum concentrations of Th17-related cytokines were measured by MultiPlex The impact of individual parameters on overall survival and progression-free survival was assessed using multivariate Cox models Results: High baseline IL-17A serum concentrations were significantly associated with shorter progression-free survival [p = 0.043] Other baseline serum Th17-related cytokines and polymorphisms of IL-17 were not associated with overall survival or progression-free survival Conclusions: In this ancillary study, baseline serum IL-17A concentration is the only Th17/IL-17 related factor that was significantly associated with the response of patients with metastatic colorectal cancer to bevacizumab But this main significant result is highly dependent on one case which, if left out, weakens the data Other clinical studies are required to confirm this association Trial registration: NCT00489697 June 20, 2007 Keywords: Bevacizumab, Vascular endothelial growth factor, Th17-related cytokines, IL-17 polymorphisms, Metastatic colorectal cancer, Survival analysis: score * Correspondence: william.raoul@univ-tours.fr Equal contributors Universitộ Franỗois-Rabelais de Tours, CNRS, GICC UMR 7292, UFR de médecine, BP 3223, 10, boulevard Tonnellé, 37032 Tours Cedex 01, France Full list of author information is available at the end of the article © The Author(s) 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Lereclus et al BMC Cancer (2017) 17:220 Background Colorectal cancer is a major public health issue due to its frequency and its severity [1] It is a major cause of death in the world [2] The therapeutic arsenal against metastatic colorectal cancer was strengthened by the addition of monoclonal antibodies to chemotherapy Bevacizumab (Avastin® [3]) is a humanized IgG1 that binds to the vascular endothelial growth factor (VEGF), a well known pro-angiogenic growth factor [4] which favors tumors and metastasis Bevacizumab is widely used in the treatment of patients with advanced colorectal cancer [5] Though bevacizumab prolongs survival of patients with metastatic colorectal cancer, some individuals not respond to treatment [6] and it is difficult to identify at an early stage who will benefit or not from this biopharmaceutical Our team has recently showed that antigenic burden influences bevacizumab pharmacokinetics in patients with metastatic colorectal cancer [7] In this study, bevacizumab pharmacokinetics was also influenced by baseline VEGF and CarcinoEmbryonic Antigen (CEA) concentrations and by the number of extra-hepatic metastases These differences in bevacizumab pharmacokinetics between patients are relevant since bevacizumab concentrations are associated with progression-free (PFS) and overall survival (OS) of patients [7] There is however a need to identify other early biomarkers that could be predictive of response to anti-VEGF biopharmaceuticals Several studies suggest that interleukin-17 (IL-17A or IL-17 or cytotoxic T-lymphocyte-associated protein 8) plays a major role in colorectal cancer progression [8–12] and in the resistance to anti-VEGF treatment in murine models [13] or chemotherapy [14, 15] IL-17A is a proinflammatory cytokine that contributes to the pathogenesis of inflammatory and auto-immune diseases [16, 17] but that also seems to be highly associated with cancer progression [18, 19] A major source of IL-17 is a lineage of T cells known as CD4+ T helper 17 cells (Th17 cells) which differ from the Th1 and Th2 subsets [20] IL-17 is also secreted by other cell types of the immune system including lymphocytes NKT-17, γδT-17, CD8+ Tc17, polymorphonuclear neutrophils and intestinal Paneth cells [21, 22] IL-17 homodimers signal through the complex formed by heterodimer IL-17 Receptor (R)A/IL-17RC Interleukin-17 F (IL-17F) is a recently described member of the IL-17 family with a great homology to IL-17A IL-17F is mainly secreted by T CD4+ and γδT-17 lymphocytes and acts as homodimers or heterodimers with IL-17A [23] It signals through the same receptors as IL-17A, with a better affinity to IL-17RC However, its role and function in inflammation and cancer, a priori close to those of IL-17A, need to be further investigated In colon cancer, these two cytokines could have in fact opposite effects since IL-17A favors cancer development and IL-17F appears to be a protective factor Page of against tumorigenesis [18, 24] Notably, IL-17A polymorphisms were associated with increased risk of colorectal cancer development when comparing patients to healthy controls in Iranian and Tunisian populations [25, 26] Interestingly IL-17F polymorphisms was observed to be mainly protective in the same populations [25, 27] To our knowledge, there is no data concerning serum Th17-related cytokines concentration or Il-17-related polymorphisms that may be predictive of the clinical impact of bevacizumab in metastatic colorectal cancer or be at least implicated in the prognosis of the disease, in this particular treatment schedule The objective of the present study was therefore to evaluate the association of individual sources of variability related to IL-17 pathway with OS and PFS of patients with metastatic colorectal cancer treated with a bevacizumab-based regimen In addition to biomarkers previously assessed, i.e CEA, VEGF and bevacizumab concentrations [7], we studied the influence of baseline serum Th17-related cytokines concentrations (before bevacizumab treatment initiation) and of selected IL-17A (G197A, rs2275913) and IL-17F (T7488C, rs763780) polymorphisms on clinical outcomes Methods Study design This ancillary study is part of a French multicenter non-comparative, prospective, open-label, observational study (NCT00489697, registration first received June 20, 2007) The study is also registered as ID number INCA06FT/STIC-AVASTIN The main study was designed to evaluate the usefulness of hepatic contrast-enhanced ultrasound sonography as predictor of response to bevacizumab-based chemotherapy in patients with metastatic colorectal cancer Briefly, patients enrolled between January 2007 and December 2010 received mg/kg bevacizumab intravenously with two-week intervals in combination with chemotherapy Tumor response was assessed by RECIST criteria (response evaluation criteria in solid tumors [28]) using spiral Computed Tomography (CT) before treatment and at day 60 after the beginning of the treatment The end of follow-up was December 2012 Finally, 122 patients received at least the four bevacizumab infusions This study was designed in accordance with legal requirements and the Declaration of Helsinki and was approved by the ethics committee of Tours University Hospital, France All patients gave written informed consent to participate in this study, including constitutional genetic analyses Eligible patients (18–80 years old) had histologically confirmed metastatic colorectal cancer with at least one hepatic metastasis detected by ultrasonography, a life expectancy of more than two months, a World Health Organization performance status of two or Lereclus et al BMC Cancer (2017) 17:220 less and were mostly treated as first line treatment with a bevacizumab-based chemotherapy Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) Genomic DNA was extracted from peripheral blood leukocytes Patients were genotyped using specific primers 100 ng/μl DNA was amplified with Taq Polymerase solution (Life Technologies, Saint-Aubin, France), including MgCl2 (2.5 mM) and dNTP (5 nM) PCR reactions were performed in 35 cycles of denaturation at 95 °C for s and primer annealing and extension at 60 °C for 45 s IL-17A rs2275913 (G197A) sequence was amplified with the following primers previously validated by Wu et al [29]: Forward 5′-AAC AAG TAA GAA TGA AAA GAG GAC ATG GT-3′ Reverse 5′-CCC CCA ATG AGG TCA TAG AAG AAT C-3′ Il-17F rs763780 (A7488G) was amplified with the following primers previously validated by Wrobel et al [30]: Forward 5′-ACC AAG GCT GCT CTG TTT CT-3′ Reverse 5′-GGT AAG GAG TGG CAT TTC TA-3′ PCR products were digested overnight at 37 °C with FastDigest XagI (EcoNI, Fermentas, Illkirch, France) for rs2275913 variant and with FastDigest NlaIII (Hin1II, Fermentas) for rs763780 variant The fragments were then separated on 8% TBE polyacrylamide gels (Invitrogen) and visualized after staining with ethidium bromide under UV light The visualized DNA products for rs2275913 were 102 bp (AA genotype), 102 + 68 + 34 bp (AG genotype) and 68 + 34 bp (GG genotype) The visualized products for rs763780 were 412 bp (GG genotype), 412 + 288 + 124 bp (AG genotype) and 288 + 124 bp (AA genotype) Page of cytokines concentrations (IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IFNγ, TNFα and sCD40L) and gene polymorphisms (IL-17A, GG vs AA + AG carriers; IL-17F, AA vs AG) as covariates of Cox models The association between these covariates and OS or PFS, was assessed using forward and backward stepwise procedures The relationship between survival and the following variables was also examined: age at inclusion, gender, bevacizumab trough concentrations before the second injection (Ctrough;